Microscale Nucleic Acid Synthesis for Template-Free Genome Assembly
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Solution Overview
Problem
Existing nucleic acid synthesis methods, particularly for large de novo molecules, are limited by the requirement of a template and lack efficiency in producing complex nucleic acid molecules such as plasmids, chromosomes, and genomes.
Innovation Solution
The use of multiwell plates with magnetic beads and electrochemically generated acid (EGA) in each well, along with other reagents, allows for non-template directed synthesis of nucleic acid molecules, enabling the assembly of smaller molecules into larger structures like plasmids and genomes, with error correction processes and automated control of reagents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If template-directed PCR is used to generate nucleic acid molecules, then the process is simple and efficient, but it requires template nucleic acid which limits de novo synthesis capabilities
Solution Approach 1:
The patent segments the synthesis process into two distinct phases: (1) de novo synthesis of short oligonucleotide segments without template using chemical synthesis methods, and (2) assembly of these segments into longer nucleic acid molecules using PCR with the segments as templates. This segmentation allows each phase to use the most appropriate method for its specific requirement, resolving the contradiction between simplicity and de novo capability.
Solution Approach 2:
The patent performs preliminary chemical synthesis of short oligonucleotide segments before the PCR assembly step. These pre-synthesized segments serve as both the starting materials and templates for subsequent PCR reactions. This preliminary action enables de novo synthesis of long nucleic acid molecules by breaking down the complex task into manageable preparatory steps followed by amplification.
2Ease of manufacture
If chemical synthesis is used for short nucleic acid molecules, then the process is straightforward, but it cannot generate large de novo nucleic acid molecules
Solution Approach 1:
The patent divides the nucleic acid molecule construction into segments: chemical synthesis produces short oligonucleotide segments (typically 50-200 bases), which are then assembled into longer molecules through PCR. This segmentation allows chemical synthesis to operate within its optimal size range while achieving overall large molecule construction through iterative assembly.
Solution Approach 2:
The patent employs a nested assembly strategy where short chemically-synthesized oligonucleotide segments are nested together through overlapping regions and PCR amplification to form progressively longer nucleic acid molecules. Each segment contains overlapping sequences that serve as priming sites for extension, allowing nested assembly from small to large structures.
3Productivity
If traditional PCR is used to amplify nucleic acid, then amplification is efficient, but template nucleic acid is required which limits applications
Solution Approach 1:
The patent performs preliminary chemical synthesis of oligonucleotide segments that contain the desired sequence information, eliminating the need for pre-existing template DNA. These chemically-synthesized segments serve as the starting material for PCR, allowing amplification of sequences that did not previously exist in nature or in the system.
Solution Approach 2:
The patent segments the template requirement into manageable oligonucleotide pieces that can be chemically synthesized. Rather than requiring a complete template molecule for PCR, the system uses segmented oligonucleotides with overlapping regions that provide sufficient template information for amplification of the full-length product.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method facilitates the efficient production of large nucleic acid molecules with high sequence fidelity, enabling the synthesis of complex molecules like plasmids, chromosomes, and genomes, and their assembly into self-replicating forms, suitable for various biological pathways and cellular applications.
Implementation Method 1
the plate comprises a bead (e.g., a magnetic bead) located in each of a plurality of wells of the plate
Implementation Method 2
an electrochemically generated acid (EGA) being present in one or more of the plurality of wells
Data Source
AI summary
The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).